| 1. |
- Stenler, S., et al.
(författare)
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Micro-minicircle gene therapy : Implications of size on fermentation, complexation, shearing resistance, and expression
- 2014
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 3, s. e140-
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Tidskriftsartikel (refereegranskat)abstract
- The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, supercoiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.
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| 2. |
- Fredriksson, A., et al.
(författare)
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Labeling of human C-peptide by conjugation with N-succinimidyl-4- F-18 fluorobenzoate
- 2001
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Ingår i: Journal of labelled compounds & radiopharmaceuticals. - : Wiley. - 0362-4803 .- 1099-1344. ; 44:7, s. 509-519
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Tidskriftsartikel (refereegranskat)abstract
- We have labeled proinsulin connecting peptide (C-peptide) with fluorine-18 (t(1/2) = 109.7min) in order to perform in vivo biodistribution and pharmacokinetic studies with position emission tomography (PET). This study reports the optimization of the conjugation labeling in the N-terminal with N-succinimidyl-4-[F-18]fluorobenzoate ([F-18]SFB). In preparative runs N-4-[F-18]fluorobenzoyl-C-peptide ([F-18]FB-C-peptide) was produced in 8-12% decay-corrected yields, counted from resolubilized [F-18]F-, in less than 5h. The specific radioactivity of [F-18]FB-C-peptide, determined using ELISA for one of the preparations, was around 70 GBq/mu mol at end of synthesis.
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| 3. |
- Jonasson, P., et al.
(författare)
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Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.
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